trans Arachidonic acid isomers inhibit NADPH-oxidase activity by direct interaction with enzyme components.

NADPH-oxidase is an enzyme that represents, when activated, the major source of non-mitochondrial reactive oxygen species. In phagocytes, this production is an indispensable event for the destruction of engulfed pathogens. The functional NADPH-oxidase complex consists of a catalytic membrane flavocytochrome b (Cytb(558)) and four cytosolic proteins p47(phox), p67(phox), Rac and p40(phox). The NADPH-oxidase activity is finely regulated spatially and temporally by cellular signaling events that trigger the translocation of the cytosolic subunits to its membrane partner involving post-translational modifications and activation by second messengers such as arachidonic acid (AA). Arachidonic acid in its natural cis-poly unsaturated form (C20:4) has been described to be an efficient activator of the enzyme in vivo and in vitro. In this work, we examined in a cell-free system whether a change of the natural cis geometry to the trans configuration, which could occur either by diet or be produced by the action of free radicals, may have consequences on the functioning of NADPH-oxidase. We showed the inability of mono-trans AA isomers to activate the NADPH-oxidase complex and demonstrated the inhibitory effect on the cis-AA-induced NADPH oxidase activation. The inhibition is mediated by a direct effect of the mono-trans AA which targets both the membrane fraction containing the cytb(558) and the cytosolic p67(phox). Our results suggest that the loss of the natural geometric feature (cis-AA) induces substantial structural modifications of p67(phox) that prevent its translocation to the complex.

Key role of proline L209 in connecting the distant quinone pockets in the reaction center of Rhodobacter sphaeroides.

Photosynthetic bacterial reaction centers convert light excitation into chemical free energy. The initial electron transfer leads to the consecutive semireductions of the primary (Q(A)) and secondary (Q(B)) quinone acceptors. The Q(A)(-) and Q(B)(-) formations induce proton uptake from the bulk. Their magnitudes (H(+)/Q(A)(-) and H(+)/Q(B)(-), respectively) probe the electrostatic interactions within the complex. The pH dependence of H(+)/Q(A)(-) and H(+)/Q(B)(-) were studied in five single mutants modified at the L209 site (L209P-->F,Y,W,E,T). This residue is situated at the border of a continuous chain of water molecules connecting Q(B) to the bulk. In the wild type (WT), a proton uptake band is present at high pH in the H(+)/Q(A)(-) and H(+)/Q(B)(-) curves and is commonly attributed to a cluster of acidic groups situated nearby Q(B). In the H(+)/Q(A)(-) curves of the L209 variants, this band is systematically absent but remains in the H(+)/Q(B)(-) curves. Moreover, notable increase of H(+)/Q(B)(-) is observed in the L209 mutants at neutral pH as compared with the WT. The large effects observed in all L209 mutants are not associated with significant structural changes (Kuglstatter, A., Ermler, U., Michel, H., Baciou, L. & Fritzsch, G. Biochemistry (2001) 40, 4253-4260). Our data suggest that, in the L209 mutants, the Q(B) cluster does not respond to the Q(A)(-) formation as observed in the WT. We propose that, in the mutants, removal of the rigid proline L209 breaks a necessary hydrogen bonding connection between the quinone sites. These findings suggest an important role for structural rigidity in ensuring a functional interaction between quinone binding sites.

Revealing the involvement of extended hydrogen bond networks in the cooperative function between distant sites in bacterial reaction centers.

In reaction center proteins of photosynthetic bacteria, the amplitude of proton uptake induced by the one-electron reduction of either of the two quinone electron acceptors (Q(A) and Q(B)) is an intrinsic observable of the electrostatic interactions associated with the redox function of the complex. We report here that, in Rhodobacter capsulatus, complete restoration of proton uptake (upon formation of Q(A)(-) and Q(B)(-)) to the level found in the wild type is observed in a mutant reaction center in which a tyrosine substitution in the Q(A) environment (Ala(M274) --> Tyr) is coupled with mutations of acidic residues near Q(B) (Glu(L212) --> Ala/Asp(L213) --> Ala) that initially cancel the proton uptake above pH 8. This result demonstrates that proton uptake occurs by strong cooperation between structural motifs, such as hydrogen-bonded networks, that span the 18 A distance between the two quinone acceptors.

X-ray structure analyses of photosynthetic reaction center variants from Rhodobacter sphaeroides: structural changes induced by point mutations at position L209 modulate electron and proton transfer.

The structures of the reaction center variants Pro L209 --> Tyr, Pro L209 --> Phe, and Pro L209 --> Glu from the photosynthetic purple bacterium Rhodobacter sphaeroides have been determined by X-ray crystallography to 2.6-2.8 A resolution. These variants were constructed to interrupt a chain of tightly bound water molecules that was assumed to facilitate proton transfer from the cytoplasm to the secondary quinone Q(B) [Baciou, L., and Michel, H. (1995) Biochemistry 34, 7967-7972]. However, the amino acid exchanges Pro L209 --> Tyr and Pro L209 --> Phe do not interrupt the water chain. Both aromatic side chains are oriented away from this water chain and interact with three surrounding polar side chains (Asp L213, Thr L226, and Glu H173) which are displaced by up to 2.6 A. The conformational changes induced by the bulky aromatic rings of Tyr L209 and Phe L209 lead to unexpected displacements of Q(B) compared to the wild-type protein. In the structure of the Pro L209 --> Tyr variant, Q(B) is shifted by approximately 4 A and is now located at a position similar to that reported for the wild-type reaction center after illumination [Stowell, M. H. B., et al. (1997) Science 276, 812-816]. In the Pro L209 --> Phe variant, the electron density map reveals an intermediate Q(B) position between the binding sites of the wild-type protein in the dark and the Pro L209 --> Tyr protein. In the Pro L209 --> Glu reaction center, the carboxylic side chain of Glu L209 is located within the water chain, and the binding site of Q(B) remains unchanged compared to the wild-type structure.

The natural product capsaicin inhibits photosynthetic electron transport at the reducing side of photosystem II and purple bacterial reaction center: structural details of capsaicin binding.

Capsaicin, a natural quinone analog, was found to block electron transport, in both plant photosystem II (PSII) and bacterial reaction center (RC) from Rhodobacter sphaeroides, at the QB site. The mode of action of capsaicin was investigated by O2 evolution measurements and fluoresence induction studies in the case of PSII, and flash-induced absorbance spectroscopy in the case of the bacterial RC. Structural details of capsaicin binding to the bacterial RC complex were determined by X-ray crystallographic analysis.

Modeling the effects of mutations on the free energy of the first electron transfer from QA- to QB in photosynthetic reaction centers.

Numerical calculations of the free energy of the first electron transfer in genetically modified reaction centers from Rhodobacter (Rb.) sphaeroides and Rb. capsulatus were carried out from pH 5 to 11. The multiconformation continuum electrostatics (MCCE) method allows side chain, ligand, and water reorientation to be embedded in the calculations of the Boltzmann distribution of cofactor and amino acid ionization states. The mutation sites whose effects have been modeled are L212 and L213 (the L polypeptide) and two in the M polypeptide, M43(44) and M231(233) in Rb. capsulatus (Rb. sphaeroides). The results of the calculations were compared to the experimental data, and very good agreement was found especially at neutral pH. Each mutation removes or introduces ionizable residues, but the protein maintains a net charge close to that in native RCs through ionization changes in nearby residues. This reduces the effect of mutation and makes the changes in state free energy smaller than would be found in a rigid protein. The state energy of QA-QB and QAQB- states have contributions from interactions among the residues as well as with the quinone which is ionized. For example, removing L213Asp, located in the QB pocket, predominantly changes the free energy of the QA-QB state, where the Asp is ionized in native RCs rather than the QAQB- state, where it is neutral. Side chain, hydroxyl, and water rearrangements due to each of the mutations have also been calculated showing water occupancy changes during the QA- to QB electron transfer.

In Rhodobacter sphaeroides reaction centers, mutation of proline L209 to aromatic residues in the vicinity of a water channel alters the dynamic coupling between electron and proton transfer processes.

The X-ray crystallographic structure of the photosynthetic reaction center from Rhodobacter sphaeroides obtained at high resolution has revealed a number of internal water molecules (Ermler, U., Fritzsch, G., Buchanan, S. K., and Michel, H. (1994) Structure 2, 925-936; Stowell, M. H. B., McPhillips, T. M., Rees, D. C., Soltis, S. M., Abresch, E., and Feher, G. (1997) Science 276, 812-816). Some of them are organized into distinct hydrogen-bonded water chains that connect Q(B) (the terminal quinone electron acceptor of the reaction center) to the aqueous phase. To investigate the role of the water chains in the proton conduction process, proline L209, located immediately adjacent to a water chain, was mutated to the following residues: F, Y, W, E, and T. We have first analyzed the effects of the mutations on the kinetic and thermodynamic properties of the rate constants of the second electron transfer (k(AB)(2)) and of the coupled proton uptake (k(H)+) at the second flash. In all aromatic mutants, k(AB)(2) and k(H)+ are notably and concomitantly decreased compared to the wild-type, while no effect is observed in the other mutants. The temperature dependence of these rates shows activation energy values (DeltaH) similar for the proton and electron-transfer processes in the wild-type and in most of the mutants, except for the L209PW and L209PF mutants. The analysis of the enthalpy factors related to the electron and proton-transfer processes in the L209PF and the L209PW mutants allows to distinguish the respective effects of the mutations for both transfer reactions. It is noteworthy that in the aromatic mutants a substantial increase of the free energies of activation is observed (DeltaG(L209PY) < DeltaG(L209PF) < DeltaG(L209PW)) for both proton and electron-transfer reactions, while in the other mutants, DeltaG is not affected. The salt concentration dependence of k(AB)(2) shows, in the L209PF and L209PW mutants, a higher screening of the protein surface potential experienced by Q(B). Our data suggest that residues F and W in position L209 increase the polarizability of the internal water molecules and polar residues by altering the organization of the hydrogen-bond network. We have also analyzed the rates of the first electron-transfer reaction (k(AB)(1)), in the 100 micros time domain. These kinetics have previously been shown to reflect protein relaxation events possibly including proton uptake events (Tiede, D. M., Vazquez, J., Cordova, J., and Marone, P. M. (1996) Biochemistry 35, 10763-10775). Interestingly, in the L209PF and L209PW mutants, k(AB)(1) is notably decreased in comparison to the wild type and the other mutants, in a similar way as k(AB)(2) and k(H)+. Our data imply that the dynamic organization of this web is tightly coupled to the electron transfer process that is kinetically limited by protonation events and/or conformational rearrangements within the protein.

Interruption of the water chain in the reaction center from Rhodobacter sphaeroides reduces the rates of the proton uptake and of the second electron transfer to QB.

A chain of bound water molecules was recently identified in the photosynthetic reaction center (RC) from Rhodobacter sphaeroides by X-ray crystallography [Ermler et al. (1994) Structure 2, 925-936]. The possible role of the chain in proton transfer from the solution to the secondary quinone (QB) was investigated by site-directed mutagenesis and flash-induced absorbance spectroscopy. Pro L209, situated along the water chain about 9 A from QB, was changed into the aromatic residues Phe and Tyr in order to interrupt the chain. In the PL209Y (Pro L209-->Tyr) mutant, the very small changes in the QA-QB<==>QAQB- equilibrium constant (K2) and the first electron-transfer rates (kAB(1)) indicate that the mutation does not lead to large structural changes. In the PL209F (Pro L209-->Phe) mutant, a 7-fold decrease of kAB(1) is observed. It follows a pH dependence parallel to that of the wild type. It is consistent with no modification of the pK of the Glu L212 determined from the pH dependence of K2. The decreased kAB(1) may reflect some slight structural modification in this mutant and/or rearrangement of the cluster of charged residues close to the L209 position. The major effect of the mutations observed is a concomitant decrease of the rates of the second electron transfer, kAB(2), and of the proton uptake upon the second flash. The relative decrease of the kAB(2) rate values in the mutants is more pronounced above pH 8. Our results indicate that the mutations have specifically altered the pathway of proton transfer to QB.(ABSTRACT TRUNCATED AT 250 WORDS)

Proton conduction within the reaction centers of Rhodobacter capsulatus: the electrostatic role of the protein.

Light-induced charge separation in the photosynthetic reaction center results in delivery of two electrons and two protons to the terminal quinone acceptor QB. In this paper, we have used flash-induced absorbance spectroscopy to study three strains that share identical amino acid sequences in the QB binding site, all of which lack the protonatable amino acids Glu-L212 and Asp-L213. These strains are the photosynthetically incompetent site-specific mutant Glu-L212/Asp-L213-->Ala-L212/Ala-L213 and two different photocompetent derivatives that carry both alanine substitutions and an intergenic suppressor mutation located far from QB (class 3 strain, Ala-Ala + Arg-M231-->Leu; class 4 strain, Ala-Ala + Asn-M43-->Asp). At pH 8 in the double mutant, we observe a concomitant decrease of nearly 4 orders of magnitude in the rate constants of second electron and proton transfer to QB compared to the wild type. Surprisingly, these rates are increased to about the same extent in both types of suppressor strains but remain > 2 orders of magnitude smaller than those of the wild type. In the double mutant, at pH 8, the loss of Asp-L213 and Glu-L212 leads to a substantial stabilization (> or = 60 meV) of the semiquinone energy level. Both types of compensatory mutations partially restore, to nearly the same level, the original free energy difference for electron transfer from primary quinone QA to QB. The pH dependence of the electron and proton transfer processes in the double-mutant and the suppressor strains suggests that when reaction centers of the double mutant are shifted to lower pH (1.5-2 units), they function like those of the suppressor strains at physiological pH. Our data suggest that the main effect of the compensatory mutations is to partially restore the negative electrostatic environment of QB and to increase an apparent "functional" pK of the system for efficient proton transfer to the active site. This emphasizes the role of the protein in tuning the electrostatic environment of its cofactors and highlights the possible long-range electrostatic effects.

Study of wild type and genetically modified reaction centers from Rhodobacter capsulatus: structural comparison with Rhodopseudomonas viridis and Rhodobacter sphaeroides.

Reaction centers from the purple bacterium Rhodobacter (Rb.) capsulatus and from two mutants ThrL226-->Ala and IleL229-->Ser, modified in the binding protein pocket of the secondary quinone acceptor (QB), have been studied by flash-induced absorbance spectroscopy. In ThrL226-->Ala, the binding affinities for endogenous QB (ubiquinone 10) and UQ6 are found to be two to three times as high as the wild type. In contrast, in IleL229-->Ser, the binding affinity for UQ6 is decreased about three times compared to the wild type. In ThrL226-->Ala, a markedly increased sensitivity (approximately 30 times) to o-phenanthroline is observed. In Rhodopseudomonas viridis, where Ala is naturally in position L226, the sensitivity to o-phenanthroline is close to that observed in ThrL226-->Ala. We propose that the presence of Ala in position L226 is responsible for the high sensitivity to that inhibitor. The pH dependencies of the rate constants of P+QB- (kBP) charge recombination kinetics (P is a dimer of bacteriochlorophyll, and QB is the secondary quinone electron acceptor) show destabilization of QB- in ThrL226-->Ala and IleL229-->Ser, compared to the wild type. At low pH, similar apparent pK values of protonation of amino acids around QB- are measured in the wild type and the mutants. In contrast to Rb. sphaeroides, in the wild type Rb. capsulatus, kBP substantially increases in the pH range 7-10. This may reflect some differences in the respective structures of both strains or, alternatively, may be due to deprotonation of TyrL 215 in Rb. capsulatus. At pH 7, measurements of the rate constant of QA to QB electron transfer reveal a threefold greater rate in the reaction centers from wild type Rb. capsulatus (65 +/- 1 0 ps)-1 compared to Rb. sphaeroides.We suggest that this may arise from a 0.7-A smaller distance between the quinones in the former strain. Our spectroscopic data on the wild type Rb. capsulatus reaction center suggest the existence of notable differences with the Rb. sphaeroides reaction center structure.

In bacterial reaction centers protons can diffuse to the secondary quinone by alternative pathways.

The mechanisms of proton conduction to the reduced secondary quinone in bacterial reaction centers were studied in wild-type and genetically modified reaction centers from Rhodobacter capsulatus. In the L212-213AA double mutant (L212Glu----Ala, L213Asp----Ala), reaction center function is severely altered. However, a photocompetent revertant of this strain which carries a third 'compensating' mutation, M231Arg----Leu, at about 15 A from the secondary quinone, displays the normal proton binding function of the reaction center. Furthermore, the apparent pK values of group(s) involved in the stabilization of the semiquinone anion are restored by that mutation. We conclude that L212Glu and L213Asp are not obligatory residues for proton donation to QB in Rb. capsulatus. We suggest that protons can be delivered to the QB site from the cytoplasm via a network of proton channels activated by compensatory mutations, possibly involving water molecules bound in the interior of the reaction center.

Study of QB- stabilization in herbicide-resistant mutants from the purple bacterium Rhodopseudomonas viridis.

The pH dependences of the rate constants of P+QB- (kBP) and P+QA- (kAP) charge recombination decays have been studied by flash-induced absorbance change technique, in chromatophores of three herbicide-resistant mutants from Rhodopseudomonas (Rps.) viridis, and compared to the wild type. P, QA, and QB are the primary electron donor and the primary and the secondary quinone acceptors, respectively. The triazine resistant mutants T1 (Arg L217----His and Ser L223----Ala), T3 (Phe L216----Ser and Val M263----Phe), and T4 (Tyr L222----Phe), all mutated in the QB binding pocket of the reaction center, have previously been characterized (Sinning, I., Michel, H., Mathis, P., & Rutherford, A. W. (1989) Biochemistry 28, 5544-5553). The pH dependence curves of kBP in T4 and the wild type are very close. This confirms that the sensitivity toward DCMU of T4 is mainly due to a structural rearrangement in the QB pocket rather than to a change in the charge distribution in this part of the protein. In T3, a 6-fold increase of kAP is observed (kAP = 4200 +/- 300 s-1 at pH 8) compared to that of the wild type (kAP = 720 +/- 50 s-1 at pH 8). We propose that the Val M263----Phe mutation induces a free energy decrease between P+QA- and P+I- (delta G zero IA) (I is the primary electron acceptor) of about 49 meV. The very different pH dependence of kAP in T3 suggests a substantial change in the QA pocket. The 2.5 times increase of kAP above pH 9.5 in the wild type is no longer detected in T3.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of the protein-protein interactions in the thermodynamics of the electron-transfer process in the reaction centers from Rhodopseudomonas viridis.

Reaction centers from Rhodopseudomonas viridis were reconstituted into dimyristoylphosphatidylcholine (DMPC) and dielaidoylphosphatidylcholine (DEPC) liposomes. Freeze-fracture electron micrographs were performed on the samples frozen from temperatures above and below the phase transition temperatures of those lipids (Tc = 23 and 9.5 degrees C, in DMPC and DEPC, respectively). Above Tc, in the fluid conformation of the lipids, the reaction centers are randomly distributed in the vesicle membranes. Below Tc, aggregation of the proteins occurs. The Arrhenius plots of the rate constants of the charge recombination between P+ and QA- display a break at about 24 degrees C in DMPC vesicles and about 10 degrees C in DEPC vesicles (P represents the primary electron donor, a dimer of bacteriochlorophyll, and QA the primary quinone electron acceptor). This is in contrast to what was previously observed for the proteoliposomes of egg yolk phosphatidylcholine and for chromatophores [Baciou, L., Rivas, E., & Sebban, P. (1990) Biochemistry 29, 2966-2976], for which Arrhenius plots were linear. In DMPC and DEPC proteoliposomes, the activation parameters were very different on the two sides of Tc (delta H degrees for T less than Tc = 2.5 times delta H degrees for T greater than Tc), leading however, to the same delta G degrees values. Taking into account the structural and thermodynamic data, we suggest that, in vivo, protein-protein interactions play a role in the thermodynamic parameters associated with the energy stabilization process within the reaction centers.

P+QA- and P+QB- charge recombinations in Rhodopseudomonas viridis chromatophores and in reaction centers reconstituted in phosphatidylcholine liposomes. Existence of two conformational states of the reaction centers and effects of pH and o-phenanthroline.

The P+QA- and P+QB- charge recombination decay kinetics were studied in reaction centers from Rhodopseudomonas viridis reconstituted in phosphatidylcholine bilayer vesicles (proteoliposomes) and in chromatophores. P represents the primary electron donor, a dimer of bacteriochlorophyll; QA and QB are the primary and secondary stable quinone electron acceptors, respectively. In agreement with recent findings for reaction centers isolated in detergent [Sebban, P., & Wraight, C.A. (1989) Biochim. Biophys. Acta 974, 54-65] the P+QA- decay kinetics were biphasic (kfast and kslow). Arrhenius plots of the kinetics were linear, in agreement with the hypothesis of a thermally activated process (probably via P+I-; I is the first electron acceptor, a bacteriopheophytin) for the P+QA- charge recombination. Similar activation free energies (delta G) for this process were found in chromatophores and in proteoliposomes. Significant pH dependences of kfast and kslow were observed in chromtophores and in proteoliposomes. In the pH range 5.5-11, the pH titration curves of kfast and kslow were interpreted in terms of the existence of three protonable groups, situated between I- and QA-, which modulate the free energy difference between P+I- and P+QA-. In proteoliposomes, a marked effect of o-phenanthroline was observed on two of the three pKs, shifting one of them by more than 2 pH units. On the basis of recent structural data, we suggest a possible interpretation for this effect, which is much smaller in Rhodobacter sphaeroides. The decay kinetics of P+QB- were also biphasic. Marked pH dependences of the rate constants and of the relative proportions of both phases were also detected for these decays. The major conclusion of this work comes from the biphasicity of the P+QB- decay kinetics. We had suggested previously that biphasicity of the P+QA- charge recombination in Rps. viridis comes from nonequilibrium between protonation states of the reaction centers due to comparable rates of the protonation events and charge recombination. This hypothesis does not hold since the P+QB- decays occur on a time scale (tau approximately 300 ms at pH 8) much longer than protonation events. This leads to the conclusion that kfast and kslow (for both P+QA- and P+QB-) are related to conformational states of the reaction centers, existing before the flash. In addition, the fast and slow decays of P+QB- are related to those measured for P+QA-, via the calculations of the QA-QB in equilibrium QAQB- apparent equilibrium constants, K2.(ABSTRACT TRUNCATED AT 400 WORDS)